Method of identifying interleukin 1β converting enzyme inhibitors

ABSTRACT

A compound of the formula 
     
         R.sup.1 --A.sup.2 --A.sup.1 --Asp-p-nitroanilide           I 
    
     wherein: 
     A 1  is a residue of any of the naturally occurring α-amino acids or a homolog, analog or derivative of a natural α-amino acid; 
     A 2  is a residue of a lipophilic α-amino acid; 
     R 1  is alkylcarbonyl, phenalkylcarbonyl, alkoxycarbonyl, phenalkoxycarbonyl, alkylaminocarbonyl, phenalkylaminocarbcnyl or R 2  --A 3  wherein 
     A 3  is a residue of a lipophilic α-amino acid; and 
     R 2  is alkylcarbonyl, alkoxycarbonyl or phenylalkoxycarbonyl, and 
     a method of detecting inhibitors of interleukin 1β converting enzyme (ICE) comprising evaluating a test compound&#39;s capacity to inhibit the ICE-induced hydrolysis of a compound of the formula I. The greater the ability of a test compound to inhibit such hydrolysis, the greater its expected activity in treating inflammation as well as diseases whose pathogenesis is induced or sustained by interleukin-1β. Also disclosed is the following intermediate, useful for synthesizing the compounds of formula I: ##STR1##

This is a division of application Ser. No. 08/354,685, filed on Dec. 12,1994, now U.S. Pat. No. 5,498,695.

BACKGROUND OF THE INVENTION

This invention is concerned with para-nitroanilide peptides and methodsof using such peptides to detect inhibitors of interleukin 1β convertingenzyme (ICE). Such inhibitors are useful in treating inflammatoryconditions in mammals, especially man.

Current therapies for arthritis are severely limited by the side effectsof available drugs and their ineffectiveness beyond treatment fordisease symptoms. The most widely used drugs are agents (thenon-steroidal antiinflammatory drugs, NSAIDS) which inhibit thecyclooxygenase pathway of arachidonic acid metabolism. While thesecompounds are effective in controlling the symptoms of arthritis, theyare not disease remittive. Furthermore, cyclooxygenase inhibition isgenerally associated with the major side-effect of NSAID therapy,gastrointestinal irritation. Steroids are used in the more severe casesof arthritis and are very effective. However, long term therapy usingsteroids is seldom tolerable. Second line antiinflammatory agents suchas gold, penicillamine, chloroquine and methotrexate are also beset withside effect issues which severely limit their general utility.

Interleukin-1 (IL-1) has been strongly implicated as a key mediator oftissue damage in osteo- and rheumatoid arthritis. Lowering levels ofIL-1 in a diseased joint would be expected to halt continueddegeneration and perhaps allow joint repair to take place. One approachto reducing levels of IL-1 is to block the generation of mature IL-1βfrom its biologically inactive precursor, pro-IL-1β, by inhibition ofthe interleukin-1β converting enzyme (ICE). This invention relates to anovel series of compounds which are substrates for ICE. The compoundsmay be used to detect ICE inhibitors which are useful for the treatmentof diseases characterized by inflammation as well as diseases whosepathogenesis is induced or sustained by interleukin-1β. Such diseasesinclude inflammatory bowel disease, psoriasis, allergic encephalitis,gingivitis, systemic lupus erythematosus, diabetes melitis, gout, septicshock and adult respiratory distress syndrome. It is expected that suchinhibitors will not elicit the side effects associated with NSAIDtherapy (due to cyclooxygenase inhibition), steroids or other treatmentscurrently in use.

SUMMARY OF THE INVENTION

The present invention relates to a compound of the formula

    R.sup.1 --A.sup.2 --A.sup.1 --Asp-p-nitroanilide           I

wherein:

A¹ is a residue of any of the naturally occurring α-amino acids (e.g.,histidine, alanine, phenylalanine, glutamic acid, lysine, or asparticacid) or a homologue, analog or derivative of a natural a-amino acid;

A² is a residue of a lipophilic α-amino acid (e.g. valine, alanine,leucine, isoleucine or phenylalanine);

R¹ is alkylcarbonyl, phenalkylcarbonyl, alkoxycarbonyl,phenalkoxycarbonyl, alkylaminocarbonyl, phenalkylaminocarbonyl or R²--A³ wherein

A³ is a residue of a lipophilic α-amino acid (e.g. tyrosine,phenylalanine, leucine, isoleucine or valine); and

R² is alkylcarbonyl, alkoxycarbonyl or phenylalkoxycarbonyl.

The following are preferred compounds of the invention:

AcTyrValAlaAsp-p-nitroanilide;

PhCH₂ CH₂ COValAlaAsp-p-nitroanilide;

PhCH₂ NHCOValAlaAsp-p-nitroanilide₃ ;

t-BOCValAlaAsp-p-nitroanilide;

AcValAlaAsp-p-nitroanilide; and

CBZValAlaAsp-p-nitroanilide.

The present invention also relates to a method of detecting inhibitorsof interleukin 1β converting enzyme (ICE) comprising evaluating acompound's capacity to inhibit the ICE-induced hydrolysis of a compoundof the formula I. The greater the ability of a compound to inhibit suchhydrolysis, the greater its expected activity in treating inflammationas well as diseases whose pathogenesis is induced or sustained byinterleukin-1β.

The abbreviations used herein to denote amino acids are well known andstandard in the art and include the following: Ala, alanine; Pro,proline; His, histidine; Cys, cysteine; Cys(Me), methylcysteine; Phe,phenylalanine; Val, valine; Ile, isoleucine; Leu, leucine; Tyr,tyrosine; Glu, glutamic acid; Lys, lysine; Asp, aspartic acid; and Val,valine.

Other abbreviations used herein include the following: FMOC,fluorenylmethyloxycarbonyl; CBZ, benzyloxycarbonyl; Ac, acetyl; Ph,phenyl; t-BOC, t-butoxycarbonyl.

Many homologues, analogues and derivatives of natural α-amino acids arereadily available, either from commercial sources or because they may beprepared by standard methods well-known to those of ordinary skill inthe art. Examples of such compounds include phenylglycine,t-butylglycine, p-chlorophenylalanine, α-methylleucine,1-aminocyclopentane-1-carboxylic acid.

The present invention also relates to the following intermediate, usefulfor synthesizing the compounds of formula I: ##STR2##

DETAILED DESCRIPTION OF THE INVENTION

The compounds of the present invention, having the formula I, as definedabove, are readily and generally prepared by the general methodsdescribed below. ##STR3##

The most preferred procedure, shown in Scheme 1, is to first couple adi- or tripepride (R¹ --A² --A¹ OH), which can be prepared by standardmethods known in the art, with the β-t-butyl ester ofaspartyl-p-nitroanilide (the product of Preparation 1). This couplingcan be induced by any number of methods known in the art such as, butnot limited to, those based on dicyclohexylcarbondiimide,1-(dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (DEC.HCl) (themethod used herein), isobutyl chloroformate, andN,N-bis[2-oxo-3-oxazolidinyl]phophorodiamidic chloride. Additives suchas, but not limited to, N-hydroxysuccinimide or N-hydroxybenzotriazole,which are typically used in such couplings can be included. The solventused for this coupling can be any reaction inert solvent such as, butnot limited to, DMF (dimethylformanide), THF (tetrahydrofuran), dioxane,and methylene chloride. The coupling reaction can be performed at fromabout -20° to about 100° C., with temperatures about 15° to about 30° C.being preferred. The second step of the preferred procedure involvescleaving the β-t-butyl ester of the aspartyl residue which is carriedout with strong acids such as, but not limited to, trifluoroacetic acid(TFA), hydrogen chloride, hydrogen bromide, p-toluenesulfonic acid, andmethanesulfonic acid. Co-solvents such as, but not limited to, methylenechloride, dioxane and ethyl acetate can be used. TFA/methylene chloridemixtures are preferred. The reaction can be performed at from about -50°to about +50° C., with about 15° to about 30° C. being preferred.Additives such as, but not limited to, anisole and thioanisole can beincluded to prevent side reactions from occurring during the cleavage ofthe ester. ##STR4##

In those cases where R¹ or R² is a t-butoxycarbonyl (t-BOC) group thepreferred procedure is to first react aspartyl-p-nitroanilidehydrochloride (the product of Example 4, Step B) with a preactivatedN-t-BOC-protected amino acid derivative in the presence of a base. Thiswill give t-BOC-A¹ -Asp-p-nitroanilide. Preactivation of thet-BOC-protected amino acid derivative may be as, but is not limited to,the N-hydroxysuccinimide or pentafluorophenyl esters. The base can be,but is not limited to, tertiary amine bases such as triethyl amine,diisopropyl ethyl amine, pyridine and N-methylmorpholine. The reactionis performed in a reaction inert solvent such as, but not limited to,DMF, THF, dioxane, and methylene chloride. The reaction can be performedat from about -20° to about 100° C. with temperatures about 15° to about30° C. being preferred. In the second step the N-terminal t-BOC group isremoved with strong acids such as, but not limited to, trifluoroaceticacid (TFA), hydrogen chloride, hydrogen bromide, p-toluenesulfonic acid,and methanesulfonic acid. Co-solvents such as, but not limited to,methylene chloride, dioxane and ethyl acetate can be used. TFA/methylenechloride mixtures are preferred. This reaction can be performed at fromabout -50° to about +50° C. with about 15° to about 30° C. beingpreferred. Co-solvents such as, but not limited to, anisole andthioanisole can be included to prevent side reactions from occurringduring removal of the t-BOC group. In a third step, the product of thisreaction is coupled as described above with a preactivatedN-t-BOC-protected amino acid derivative in the presence of a base whichwill give t-BOC-A¹ -Asp-p-nitroanilide. The two step sequence can berepeated again to give t-BOC-A³ -A² -A¹ -Asp-p-nitroanilide.

The compounds of formula 1 are substrates for ICE and, in conjunctionwith ICE that has been partially to totally purified or more preferablythat has been immobilized in an active form by adsorption onto antibodycoated protein A functionalized agarose beads, can be used for detectinginhibitors of ICE. Inhibitors of ICE may be used in treatinginflammatory diseases in which interleukin-1β plays a role. Adsorptionof ICE onto protein A functionalized agarose beads is achieved bycoating the beads with a polyclonal antibody specific to the N-terminalregion of ICE wherein the F_(c) region of the antibody binds to theprotein A. The F_(ab) portion of the antibody remains free to bind toICE which it does when the beads are then treated with a partiallypurified preparation of ICE derived from THP-1 cells. The ICE so boundretains its catalytic activity.

The following Preparations and Examples illustrate the preparation ofthe compounds of the present invention and their use in detecting ICEinhibitors. Abbreviations used below are defined either the first timethey are used or on pages 2, 4 and 6 above.

PREPARATION 1 HAsp (β-t-butyl)-p-nitroanilide

A. FMOCAsp(β-t-butyl)-p-nitroanilide

Using the procedure of Rijkers et al. (Recl. Trav. Chim Pays-Bas, 110,347 (1991)) FMOCAsp(β-t-butyl)OH (10.29 g, 25.0 mmole), p-nitroaniline(3.45 g, 25.0 mmole) and POCl₃ (4.22 g=2.56 mL, 27.5 mmole) in pyridine(75 mL) gave, after being passed through a pad of silica gel(40:60--ethyl acetate:hexane), 12.38 g (93%) of light yellow foam.Recrystallization of a portion from cyclohexene/ethyl acetate gave ananalytical sample as a light yellow powder: mp 163°-164° C. (dec. withgas evol.); ¹ H NMR (CDCl₃) δ 1.47 (s, 9H), 2.68 (dd, J=7.4, 16.8 Hz,1H), 2.97 (dd, J=3.8, 16.8 Hz, 1H), 4.24 (t, J=6.8 Hz, 1H), 4.51 (d,J=6.8 Hz, 2H), 4.6-4.7 (m, 1H), 6.0-6.15 (m, 1H), 7.25-7.35 (m, 2H),7.35-7.45 (m, 2H), 7.58 (d, J=7.4 Hz, 2H), 7.67 (d, J=9.2 Hz, 2H), 7.77(d, J=7.6 Hz, 2H, 8.21 (d, J=9.2 Hz, 2H), 9.0-9.1 (br s, 1H); MS (LSIMS)m/e 532 (11, M⁺ +1), 476 (29), 179 (100); [α]_(D) ²⁰ +39.2° (c=1.0,DMF); Analysis calculated for C₂₉ H₂₉ N₃ O₇ : C, 65.52; H, 5.50; N,7.91; found: C, 65.61; H, 5.58; N, 7.80.

B. HAsp(β-t-butyl)-p-nitroanilide

FMOCAsp(β-t-butyl)-p-nitroanilide (2.13 g, 4.0 mmole) and DBU(1,8-diazabicyclo[5.4.0]undecene-7) (609 g, 4.0 mmole) were stirredtogether in dry dimethylformanide (DMF)(40 mL) for 1 hour. The reactionmixture was then diluted with ether (200 mL) and extracted with 1N HCl(3×20 mL). The combined aqueous extracts were washed with ether and thenbasified with a calculated amount of K₂ CO₃ (8.3 g, 60 mmole). Thisaqueous solution was extracted with ether (9×50 mL). The combined etherextracts were dried over MgSO₄. Filtration and concentration gave ayellow oil consisting of the desired product, some DBU and DMF. This wastaken up in 1:1 ethyl acetate:hexane (50 mL) and poured onto a pad ofsilica gel. Elution with 1:1 ethyl acetate:hexane (7×50 mL) and ethylacetate (3×200 mL) completely eluted the product. Concentration of theappropriate fractions gave 955 mg (77%) of light yellow solid.Recrystallization from cyclohexane/ethyl acetate gave an analyticalsample as a pale yellow powder: mp 143°-144° C.; ¹ H NMR (CDCl₃) δ 1.44(s, 9H), 2.1-2.3 (brs, 2H), 2.82 (dd, J=6.7, 16.9 Hz, 1H), 2.89 (dd,J=4.4, 16.9 Hz, 1H), 3.82 (dd, J=4.4, 6.7 Hz, 1H), 7.77 (d, J=9.2 Hz,2H), 8.20 (d, J=92 Hz, 2H), 10.0-10.1 (br s, 1H); MS (LSIMS) m/e 310(43, M⁺ +1), 254 (97), 154 (100); [α]_(D) ²⁰ -4.9° (c=1.0, DMF); HPLCret. time: 2.07 min (40%), 5.56 min (50%); Analysis calculated for C₁₄H₁₉ N₃ O₅ : C,54.36; H, 6.19; N, 13.59; found C, 54.69; H, 6.22; N,13.37.

PREPARATION 2 CBZValAlaOCH₃

CBZVal N-hydroxysuccinimide ester (8.71 g, 25.0 mmole), alanine methylester hydrochloride (3.49 g, 25.0 mmole), DIEA (diisopropylethylamine)(3.23 g. 25.0 mmole) were combined in CH₂ Cl₂ (250 ml) and stirred atroom temperature for 20 hours. The reaction mixture was washed twice,each time first with saturated NaHCO₃ and then with 1N HCl, and was thendried over MgSO₄, filtered, and concentrated giving a white solid. Thiswas recrystallized from ethylacetate to give 5.49 g (65%) of fine whiteneedles. A second crop of 1.56 g (18%) of fine white needles wasobtained from the mother liquors: mp 163°-164° C.; ¹ H NMR (CDCl₃) δ0.93 (d, J=6.8 Hz, 3H), 0.98 (d, J=6.8 Hz, 3H), 1.40 (d, J=7.2 Hz, 3H),2.11 (hept, J=6.7 Hz, 1H), 3.74 (s, 3H), 4.01 (br t, 1H), 4.58 (pent,J=7.2 Hz, 1H), 5.11 (s, 2H), 5.38 (b d, 1H), 6.38 (br d, 1H), 7.3-7.4(m, 5H); MS (LSIMS) m/e 337 (100, M⁺ +1), 255(66); [α]_(D) ²⁰ -46.0°(c=1.0, methanol); Analysis calculated for C₁₇ H₂₄ N₂ O₅ : C, 60.70; H,7.19; N, 8.33; found: C, 60.70; H, 7.14; N, 8.33.

EXAMPLE 1 AcTyrValAlaAsp-p-nitroanilide

A. CBZTyr(O-t-butyl)ValAlaOCH₃

CBZValAlaOCH₃ (6.67 g, 19.8 mmole) was hydrogenated at 3 atm. over 10%Pd on carbon (700 mg) in CH₃ OH (100 mL) at room temperature. After 1hour, the catalyst was removed by filtration through a nylon filter. Thefiltrate was evaporated in vacuo giving a white solid which wasdissolved in a 1:1 mixture of CH₂ Cl₂ and DMF (200 mL). To this solutionwas added CBZTyr(O-t-butyl) N-hydroxysuccinimide ester (9.28 g, 19.8mmole). After being stirred at room temperature for 18 hours, themixture was concentrated in vacuo to remove the CH₂ Cl₂ and then water(300 mL) was added to precipitate the product. The solid was collected,washed with water and dissolved in ethyl acetate (500 mL). This solutionwas washed twice with saturated NaHCO₃ and twice with 1N HCl and driedover MgSO₄. Filtration and evaporation in vacuo gave a white solid whichwas recrystallized from cyclohexene (100 mL) ethyl acetate (70 mL)yielding 7.10 g (65%) of white fluffy solid. A second crop of 1.25 g(11%) was obtained from the mother liquors: mp 189°-190° C.; ¹ H NMR(DMSO-d₆) δ 0.85 (d, J=6.8 Hz, 3H), 0.88 (d, J=7.0 Hz, 3H), 1.2-1.35 (m,12H), 1,85-2.05 (m, 1H), 2.83 (dd, J=10.9, 13.8 Hz), 1H), 2.92 (dd,J=3.7, 13.8 Hz, 1H), 3.3-3-4 (m, 2 (partially obscured by H₂ Oabsorption)), 3.60 (s, 3H), 4.2-4.4 (m, 3H), 4.94 (s, 2H), 6.85 (d,J=8.4 Hz, 2H), 7.17 (d, J=8.4 Hz, 2h), 7.2-7,35 (m, 5H), 7.52 (d, J=8.7Hz, 1H), 7.83 (d, J=9.2 Hz, 1H), 8.45 (d, J=6.5 Hz, 1H); MS (LSIMS) m/e556 (100, M⁺ +1), 453 (31); [α]_(D) ²⁰ -35.1° (c=1.0 methanol); Analysiscalculated for C₃₀ H₄₁ N₃ O₇ : C, 64.84; H, 7.44; N, 7.56; found C,64.96; H, 7.35; N, 7.52.

B. AcTyr(O-t-butyl)ValAlaOCH₃

CBZTyr(O-t-butyl)ValAlaOCH₃ (5.55 g, 10.0 mmole) was hydrogenated at 3atm. over 10% Pd on carbon (500 mg) in CH₃ OH (100 mL) at roomtemperature. After 1 hour, the catalyst was removed by filtrationthrough a nylon filter. The filtrate was evaporated in vacuo giving anoil which was dissolved in THF (100 mL). To this solution was added DIEA(1.55 g, 12 mmole) and acetyl chloride (942 mg, 12 mmole). After beingstirred at room temperature overnight, the reaction mixture wasconcentrated in vacuo and the residue dissolved in CHCl₃. This solutionwas washed with 1N HCl, and then with saturated NaHCO₃ and then driedover MgSO₄. Filtration and evaporation in vacuo gave a gel-like solidwhich was recrystallized from ethyl acetate/CH₃ OH to give 2.86 g (62%)of a gel-like solid which was dried under high vacuum. A second crop of1.42 g (31%) was obtained from the mother liquors: mp 209°-211° C.; ¹ HNMR (DMSO-d₆) δ 0.82 (d, J=6.8 Hz, 3H), 0.86 (d, J=6.8 Hz, 3H) 1.24 (s,9H), 1.27 (d, J=7.3 Hz, 3H), 1.73 (s, 3H), 1.94 (hept, J=6.8 Hz, 1H),2.66 (dd, J=10.0, 14.0Hz, 1H), 2.90 (dd, J=4.3, 14.0 Hz, 1H), 3.59 (s,3H), 4.15-4.3 (m, 2H), 4.5-4.6 (m, 1H), 6.82 (d, J=8.4 Hz, 2H), 7.12 (d,J=8.4 Hz, 2H), 7.79 (d, J=9.0 Hz, 1H), 8.06 (d, J=8.4 Hz, 1H) 8.39 (d,J=6.6 Hz, 1H); MS (LSIMS) m/e 464 (100, M⁺ +1); [α]_(D) -17.0° (c=1.0,DMF); Analysis calculated for C₂₄ H₃₇ N₃ O₆ : C, 62.18; H, 8.05; N,9.06; found: C, 62.27; H, 8.18; N, 9.00.

C. AcTyr(O-t-butyl)ValAlaOH

AcTyr(O-t-butyl)ValAlaOCH₃ (2.32 g, 5.0 mmole) was slurried in 10%aqueous CH₃ OH (50 mL) and treated with LiOH.H₂ O (1.05 g, 25.0 mmole)in one portion. The reaction mixture was stirred at room temperature for2 hours and the reaction was then quenched by the addition of an excessof sulfonic acid ion exchange resin (56 g, 125 meq of H+). After beingstirred for 15 minutes, the mixture was filtered and the resin washedthoroughly with CH₃ OH. The filtrate was concentrated in vacuo to give awhite solid which was recrystallized from ethyl acetate/CH₃ OH yielding,after drying under high vacuum, 1.96 g (87%) of a white powder: mp191°-192° C. (dec. with gas evolution); ¹ H NMR (DMSO-d₆) δ 0.82 (d,J=6.8 Hz, 3H), 0.86 (d, J=6.8 Hz, 3H), 1.25 (s, 9H), 1.27 (d, J=7.3 Hz,3H, partially obscured), 1.74 (s, 3H), 1.95 (hept, J=6.8 HZ, 1H), 2.66(dd, J=10.1, 13.9 Hz, 1H), 2.92 (dd, J=4.2, 13.9 Hz, 1H), 4.1-4.25 (m,2H), 4.5-4.6 (m, 1H), 6.82 (d, J=8.4 Hz, 2H), 7.12 (d, J=8.4 Hz, 2H),7.79 (d, J=9.0 Hz, 1H), 8.07 (d, J=8.4 Hz, 1H), 8.23 (d, J=6.9 Hz, 1H);MS (LSIMS) m/e 450 (53, M⁺ +1), 189(100); [α]_(D) ²⁰ -8.7° (c=1, DMF);Analysis calculated for calculated for C₂₃ H₃₅ N₃ O₆ : C, 61.45; H,7.85; N, 9.35; found C, 61.18; H, 8.05; N, 9.26.

D. AcTyr(O-t-butyl)ValAlaAsp(β-t-butyl)-p-nitroanilide

AcTyr(O-t-butyl)ValAlaOH (687 mg, 1.53 mmole),HAsp(β-t-butyl)-p-nitroanilide (473 mg, 1.53 mmole),N-hydroxysuccinimide (264 mg, 2.29 mmole) and DEC.HCl (352 mg, 1.84mmole) were combined in dry DMF (15 mL) and the resulting pale yellowsolution stirred at room temperature for 44 hours. The reaction mixturewas diluted with 1N HCl and the resulting precipitated solid trituratedto break all chunks of solid into a finely dispersed solid. This wasthen collected and washed with 1N HCl. The solid was resuspended inaqueous NaHCO₃, triturated for 15 minutes and collected. After washingwith water and drying under high vacuum 875 mg (77%) of a white powderwas obtained. A portion of this was recrystallized from ethylacetate/CH₃ OH to give an analytical sample: mp 234°-235° C. (dec. withgas evolution); ¹ H NMR (DMSO-d₆) δ 0.82 (t,J=7.1 Hz, 3H), 1.21 (d,J=7.1 Hz, 3H), 1.24 (s, 9H), 1.34 (s, 9H), 1.74 (s, 3H), 2.57 (dd,J=7.6, 15.9 Hz, 1H), 2.63-2.72 (m, 1H), 2.76 (dd, J=6.8, 15.9 Hz, 1H),2.92 (dd, J=0.6, 10.1 Hz, 1H), 4.1-4.2 (m, 1H), 4.2-4.3 (m, 1H), 4.5-4.6(m, 1H), 4.6-4.7 (m, 1H), 6.82 (d, J=8.4 Hz, 2H), 7.13 (d, J=8.4 Hz,2H), 7.88 (d, J=9.3 Hz, 2H), 8.08 (d, J=8.4 Hz, 1H), 8.14 (d, J=6.7 Hz,1H), 8.22 (d, J=9.3 Hz, 1H), 8.38 (d, J=7.5 Hz, 1H), 10.52 (s, 1H);MS(LSIMS) m/e 741 (32, M⁺ +1), 710 (34), 432 (53), 361 (75), 305 (100);[α]_(D) ²⁰ -16.4° (c=1, DMF); Analysis calculated for C₃₇ H₅₂ N₆ O₁₀ ;C, 59.98; H, 7.08; N, 11.35; found: C, 59.78; H, 6.90; N, 11.20.

E. AcTyrValAlaAsp-p-nitroanilide

A slurry of AcTyr(O-t-butyl)ValAlaAsp(β-t-butyl)-p-nitroanilide (148 mg,0.20 mmole) in CH₂ Cl₂ (10 mL) at 0° C. was treated with 10 mL of aprechilled mixture of TFA:anisole:thioanisole (90:5.5). The resultingsolution was stirred at 0° C. for 30 minutes and at room temperature for4 hours. The mixture was concentrated in vacuo. CH₂ Cl₂ was added to theconcentrate and the solvent evaporated in vacuo. The residue was thentriturated with ether for a few hours. The solid was collected, washedthoroughly with ether and dried under vacuum to give 109 mg (87%) of awhite powder; mp 205°-206° C. (dec.); ¹ H NMR(DMSO(dimethylsulfoxide)-d₆) δ 0.82 (t, J=7.3 Hz, 6H), 1.22 (d, J=7.2Hz, 3H), 1.74 (s, 3H), 1.9-2.0 (m, 1H), 2.5-2.7 (m, 3H), 2.86 (dd,J=0.6, 10.2 Hz, 1H), 4.1-4.2 (m, 1H), 4.2-4.3 (m, 1H), 4.45-4.55 (m,1H), 4.6-4.7 (m, 1H), 6.61 (d, J=8.4 Hz, 2H), 7.01 (d, J=8.4 Hz, 2H),7.84 (d, J=9.2 Hz, 2H), 7.88 (d, 1H, partially obscured by adjacentpeak), 8.06 (d, J=8.3 Hz, 1H), 8.12 (d, J=7.0 Hz, 1H), 8.20 (d, J=9.2Hz, 2H) 8.31 (d, J=7.5 Hz, 1H), 11.2-11.4 (br s, 1H); MS (LSIMS) m/e 651(5, M⁺ +Na), 629 (3, M⁺ +1), 491 (7), 424, (3), 376 (18), 305 (47), 178(136), 136 (100); [α]_(D) ²⁰ -18.8° (c=1.0, DMF); Analysis calculatedfor C₂₉ H₃₆ N₆ O₁₀ : C, 55.40; H, 5.77; N, 13.37: found C, 55:60; H,6.24; N, 13.49.

EXAMPLE 2 PhCH₂ CH₂ COValAlaAsp-p-nitroanilide

A. PhCH₂ CH₂ COValAlaOCH₃

CBZValAlaOCH₃ (1.35 g, 4.00 mmole) was hydrogenated at 3 atm. over 10%Pd on carbon (150 mg) in CH₃ OH (40mL) at room temperature. After 1hour, the catalyst was removed by filtration through a nylon filter. Thefiltrate was evaporated in vacuo giving a white solid which was slurredin CHCl₃ (40 mL) and treated with DIEA (620 mg, 4.8 mmole) andhydrocinnamoyl chloride (741 mg, 4.4 mmole). After 1 hour at roomtemperature, the reaction mixture was washed with 1N HCl, dried withMgSO₄ filtered and concentrated in vacuo to a white solid. This wasrecrystallized from ethylacetate to give 617 mg (48%) of white powder:mp 207°-208° C.; ¹ H NMR (DMSO-d₆) δ 0.79 (d, J=6.8 Hz, 3H), 0.84 (d,J=6.7 Hz, 3H), 1.28(d, J=7.3 Hz, 3H), 1.85-2.0 (m, 1H), 2.35-2.5 (m, 2H,partially obscured by the DMSO-d₅ peak), 2.75-2.85 (m, 2H), 3.61 (s,3H), 4.15-4.3 (m, 2H), 7.1-7.3 (m, 5H), 7.90 (d, J=9.1 Hz, 1H), 8.43 (d,J=6.7 Hz, 1H); MS (FAB) m/e 335 (88, M⁺ +1), 232 (100), 204 (53);[α]_(D).sup.° -71.7° (c=1.0, methanol); Analysis calculated for C₁₈ H₂₆N₂ O₄ : C, 64.65; H, 7.84; N, 8.38; found: C, 64.85; H, 7.62; N, 8.05.

B. PhCH₂ CH₂ COValAlaOH

By the same procedure used to prepare AcTyr(O-t-butyl)ValAlaOH, PhCH₂CH₂ COValAlaOCH₃ (508 mg, 1.52 mmole) and LiOH.OH (319 mg, 7.6 mmole) in10% aqueous CH₃ OH (15 mL) gave, after quenching with sulfonic acid ionexchange resin (17.0 g, 38 meq), 511 mg (100%) of pure product as awhite powder. Recyrstallization of a portion from ethylacetate gave ananalytical sample: mp 205°-206° C.; ¹ H NMR (DMSO-d₆) δ 0.76 (d, J=6.7Hz, 3H), 0.81 (d, J=6.8 Hz, 3H), 1.24 (d, J=7.3 Hz, 3H), 1.8-1.95 (m,1H), 2.35-2.55 (m, 2H, partially obscured by the DMSO-d₅ peak),2.75-2.85 (m, 2H), 4.1-4.25 (m, 2H), 7.1-7.3 (m, 5H), 7.84 (d, J=9.1 Hz,1H), 8.23 (d, J=6.9Hz, 1H); MS (LSIMS) m/e 321 (45, M⁺ -1), 232(29),204(11), 157(100); [α]_(D) ²⁰ -2.0° (c=1.0, DMF); Analysiscalculated for C₁₇ H₂₄ N₂ O₄ : C, 63.73; H, 7.55; N, 8.75; found: C,63.78; H, 7.30; N, 8.60.

C. PhCH₂ CH₂ COValAlaAsp(β-t-butyl)-p-nitroanilide

By the same procedure used to prepare the title compound of Example 1D,PhCH₂ CH₂ COValAlaOH (401 mg, 1.20 mmole),HAsp(β-t-butyl)-p-nitroanilide (387 mg, 1.20 mmole),N-hydroxysuccinimide (216 mg, 1.5 mmole) and DEC.HCl (288 mg, 1.88mmole) in DMF (12 mL) gave 661 mg (90%) of a tan powder. This wasrecrystallized from ethyl acetate to give 459 mg (62%) of a whitepowder; mp 222°-224° C. (dec. with gas evolution); ¹ H NMR (DMSO-d₆) δ0.77 (t, J=6.9 Hz, 6H), 1.20 (d, J=7.1 Hz, 3H), 1.34 (s, 9H), 1.85-1.95(m, 1H), 2.35-2.65 (m, 3, partially obscured by the DMSO-d₅ peak),2.7-2.85 (m, 3H), 4.1-4.15 (m, 1H), 4.2-4.3 (m, 1H), 4.6-4.7 (m, 1H),7.1-7.3 (m, 5H), 7.85-7.95 (m, 3H), 8.13 (d, J=6.8 Hz, 1H), 8.21 (d,J=9.3 Hz, 2H), 8.31 (d, J=7.6 Hz, 1H), 10.56 (s, 1H); MS (LSIMS) m/e 612(4, M⁺ +1), 556 (16), 418 (15), 325(7), 303(26), 232(100), 204(62);[α]_(D) ²⁰ -17.6° (c=1.0, DMF); Analysis calculated for C₃₁ H₄₁ N₅ O₈ :C, 60.87; H, 6.76; N, 11.45; found: C, 61.04; H, 6.59; N, 11.23.

D. PhCH₂ CH₂ COValAlaAsp-p-nitroanilide

By the same procedure used to prepare the compound of Example 1E, PhCH₂CH₂ COValAlaAsp(β-t-butyl)-p-nitroanilide (122 mg, 0.2 mmole) gave 104mg (94%) of a light tan powder: mp 206°-208° C. (dec.); ¹ H NMR(DMSO-d₆) δ 0.77 (t, J=7.0 Hz, 6H), 1.21 (d, J=7.1 Hz, 3H), 1.85-2.0 (m,1H), 2.35-2.7 (m, 3, partially obscured by the DMSO-d₅ peak), 2.7-2.85(m, 3H), 4.1-4.18 (m, 1H), 4.18-4.3 (m, 1H), 4.6-4.7 (m, 1H), 7.1-7.3(m, 5H), 7.85-7.95 (m, 3H), 8.12 (d, J=6.7 Hz, 1H), 8.21 (d, J=9.3 Hz,2H), 8.29 (d, J=7.6 Hz, 1H), 10.55 (s, 1H); MS (LSIMS) m/e 578 (10, M⁺+Na), 418 (4), 303 (26), 232 (100), 204 (89); [α]_(D) ²⁰ -21.6° (c=1.0,DMF); Analysis calculated for C₂₇ H₃₃ N₅ O₈ : C, 58.37; H, 5.99; N,12.61; found: C, 58.29; H, 5.84; N, 12.43.

EXAMPLE 3 PhCH₂ NHCOValAlaAsp-p-nitroanilide

A. PhCH₂ NHCOValAlaOCH₃

CBZValAlaOCH₃ (1.35 g, 4.00 mmole) was hydrogenated at 3 atm. over 10%Pd on carbon (150 mg) in CH₃ OH (40 mL) at room temperature. After 1hour, the catalyst was removed by filtration through a nylon filter. Thefiltrate was evaporated in vacuo giving a white solid which was slurriedin CHCl₃ (40 mL) and treated with benzyl isocyanate (586 mg, 4.4 mmole).After 1 hour at room temperature, the reaction mixture was washed threetimes with 1N HCl, dried over MgSO₄, filtered and concentrated in vacuogiving 791 mg (61%) of the desired product as a white powder.Recrystallization of a portion from ethyl acetate/CH₃ OH gave ananalytical sample: mp 227°-228° C.; ¹ H NMR (DMSO-d₆) δ 0.80 (d, J=6.8Hz, 3H), 0.86 (d, J=6.7 Hz, 3H), 1.26(d, J=7.3 Hz, 3H), 1.85-1.95 (m,1H), 3.59 (s, 3H), 4.10 (dd, J=5.9, 9.2 Hz, 1H), 4.15-4.3 (m, 3H), 6.08(d, J=9.3 Hz, 1H), 6.53 (t, J=6.0 Hz, 1H), 7.15-7.35 (m, 5H), 8.39 (d,J=6.7 Hz, 1H); MS (LSIMS) m/e 336 (100, M⁺ +1), 233 (62), 203 (76);[α]_(D) ²⁰ +6.1° (c=1.0, DMF); Analysis calculated for C₁₇ H₂₅ N₃ O₄ ;C, 60.88; H, 7.51; N, 12.53; found: C, 60.98; H, 7.30; N, 12.34.

B. PhCH₂ NHCOValAlaOH

By the same procedure used to prepare the title compound of Example 1C,PhCH₂ NHCOValAlaOCH₃ (671 mg, 2.00 mmole) and LiOH.OH (168 mg, 4.00mmole) in 10% aqueous CH₃ OH (20 mL) gave after quenching with sulfonicacid ion exchange resin (18.0 g, 40 meq) 658 mg (100%) of pure productas an off-white flaky solid. Recyrstallization of a portion from ethylacetate/CH₃ OH gave an analytical sample as very fine white crystals: mp205°-206° C.; ¹ H NMR (DMSO-d₆) δ 0.79 (d, J=6.8 Hz, 3H), 0.86 (d, J=6.7Hz, 3H), 1.25 (d, J=7.3 Hz, 3H), 1.85-2.0 (m, 1H), 4.05-4.3 (m, 4H),6.09 (d, J=9.3 Hz, 1H), 6.54 (t, J=6.0 Hz, 1H), 7.15-7.35 (m, 5H), 824(d, J=7.0 Hz, 1H); MS (LSIMS) m/e 344 (22, M⁺ +Na), 322 (83, M⁺ +1), 233(70), 205(18), 189 (100); [α]_(D) ²⁰ +15.3° (c=1.0, DMF); Analysiscalculated for C₁₆ H₂₃ N₃ O₄ : C, 59.79; H, 7.21; N, 13.08; found: C,59.87; H, 7.30; N, 12.80.

C. PhCH₂ NHCOValAlaAsp(β-t-butyl)-p-nitroanilide

By the same procedure used to prepare the title compound of Example ID,PhCH₂ NHCOValAlaOH (553 mg, 1.72 mmole), HAsp(β-t-butyl)-p-nitroanilide(532 mg, 1.72 mmole), N-hydroxysuccinimide (247 mg, 2.15 mmole) andDEC.HCl (515 mg, 2.69 mmole) in DMF (17 mL) gave 1.04 g (99%) of a lightyellow powder. This was recrystallized from ethyl acetate/CH₃ OH to give667 mg (63%) of a white powder: mp 209°-210° C. (dec. with gasevolution); ¹ H NMR (DMSO-d₆) δ 0.79 (d, J=6.8 Hz, 3H), 0.84 (d, J=6.7Hz, 3H), 1.21 (d, J=7.1 Hz, 3H), 1.34 (s, 9H), 1.85-2.0 (m, 1H), 2.55(dd, J=7.7, 16.0 Hz, 1H), 2.74 (dd, J=6.7, 16.0 Hz, 1H), 4.02 (dd,J=5.8, 8.3 Hz, 1H), 4.15-4.3 (m, 3H), 4.6-4.7 (m, 1H), 6.13 (d, J=8.4Hz, 1H), 6.56 (t, J=6.0 Hz, 1H), 7.15-7.35 (m, 5H), 7.88 (d, J=9.3 Hz,2H), 8.15-8.25 (m, 3H), 8.33 (d, J=7.6 Hz, 1H), 10.50 (s, 1H); MS(LSIMS) m/e 635 (1, M⁺ +Na), 613 (3, M⁺ +1), 557 (13), 424(12), 304(13), 286 (19), 233 (100), 205 (29), 171 (27); [α]_(D) ²⁰ -2.8° (c=1.0,DMF); Analysis calculated for C₃₀ H₄₀ N₆ O₈ : C, 58.81; H, 6.58;N,13.72; found: C, 58.92; H, 6.56; N, 13.64.

D. PhCh₂ NHCOValAlaAsp-p-nitroanilide

By the same procedure used to prepare the title compound of Example IE,PhCH₂ NHCOValAlaAsp(β-t-butyl)-p-nitroanilide (123 mg, 0.2 mmole) gave102 mg (92%) of an off-white powder: mp 207°-209° C.; ¹ H NMR (DMSO-d₆)δ 0.78 (d, J=6.8 Hz, 3H), 0.84 (d, J=6.7 Hz, 3H), 1.22 (d, J=7.1 Hz,3H), 1.85-2.0 (m, 1H), 2.60 (dd, J=7.7, 16.0 Hz, 1H), 2.78 (dd, J=6.7,16.0 Hz, 1H), 4.02 (dd, J=5.8, 8.3 Hz, 1H), 4.15-4.3 (m, 3H), 4.6-4.7(m, 1H), 6.12 (d, J=8.5 Hz, 1H), 6.56 (t, J=6.0 Hz, 1H), 7.15-7.35 (m,5H), 7.88 (d, J=9.3 Hz, 2H), 8.15-8.25 (m, 3H), 8.33 (d, J=7.6 Hz, 1H),10.47 (s, 1H), 12.45 (br s, 1H); MS (LSIMS) m/e 579 (4, M⁺ +Na), 557(15,M⁺ +1), 304 (14), 286 (20), 233 (100); [α]_(D) ²⁰ -3.5° (c=1.0, DMF);Analysis calculated for C₂₆ H₃₂ N₆₀ O₈.0.5H₂ O; C, 55.01; H, 6.22; N,14.81; found: C, 55.15, H, 6.06; N, 14.54.

EXAMPLE 4 t-BOCValAlaAsp-p-nitroanilide

A. t-BOCAsp(β-t-butyl)-p-nitroanilide

Using the procedure of Rijkers et al. (Recl. Trav. Chim Pays-Bas, 110,347 (1991)) t-BOCAsp(β-t-butyl)OH (7.65 g, 25.0 mmole), p-nitroaniline(3.45 g, 25.0 mmole) and POCl₃ (4.22 g=2.56 mL, 27.5 mmole) in pyridine(75 mL) gave, after being passed through a pad of silica gel (25:75ethyl acetate:hexane), 8.93 g (87%) of light yellow foam: ¹ H NMR(CDCl₃) δ 1.47 (s, 9H), 1.49 (s, 9H), 2.69 (dd, J=6.7, 17.0 Hz, 1H),2.91 (dd, J=4.3, 17.0 Hz, 1H), 4.59 (m, 1H), 5.90 (br d, 1H), 7.69 (d,J=9.2 Hz, 2H), 8.20 (d, J=9.2 Hz, 2H), 9.20 (br s, 1H); MS (LSIMS) m/e410 (22), 394 (9), 354 (18), 298 (100), 282 (17), 254 (26); [α]_(D) ²⁰-30.0° (c=1.0, methanol); Analysis calculated for C₁₉ H₂₇ N₃ O₇ : C,55.73; H,6.65; N, 10.26; found: C, 55.50; H, 6.41; N, 10.22.

B. HAsp-p-nitroanilide Hydrochloride

t-BOC-Asp(β-t-butyl)-p-nitroanilide (8.68 g, 21.2 mmole) was dissolvedin a mixture of dioxane (200 mL) and ethyl acetate (50 mL) and cooled to0° C. The solution was then saturated with HCl gas and stirred for onehour at 0° C. The HCl was then purged from the reaction with a stream ofN₂ and the reaction mixture concentrated to a yellow glass. This wastriturated with ether, collected and dried under high vacuum to give6.79 g (yield greater than 100%; NMR indicated some ether still present)of a light yellow powder: ¹ H NMR (DMSO-d₆) δ 2.93 (dd, J=7.3, 17.5 Hz,1H), 3.01 (dd, J=5.2, 17.5 Hz, 1H), 4.32 (m, 1H), 7.18 (d, J=9.1 Hz,2H), 8.26 (d, J=9.1 Hz, 2H); MS (LSIMS) m/e 254 (54, M⁺ +1), 239 (92),221 (100), 197 (94), 195 (76).

C. t-BOCAlaAsp-p-nitroanilide

HAsp-p-nitroanilide hydrochloride (2.90 g, 10.0 mmole), t-BOCAlaN-hydroxysuccinimide ester (2.86 g, 10.0 mmole) and DIEA (1.29 g, 10.0mmole) were combined in CH₂ Cl₂ (100 mL) and stirred for 24 hours atroom temperature. The resulting turbid solution was washed twice with0.1N HCl and then dried over MgSO₄. Filtration and concentration invacuo gave a yellow foam that was chromatographed (5:30:65--aceticacid:ethyl acetate:hexane to 5:35:60--acetic acid:ethylacetate:hexane)to give 3.03 g (71%) of a light yellow foam: mp 74°-80° C.; ¹ H NMR(CDCl₃) δ 1.39 (s, 9H), 1.44 (d, J=7.2 Hz, 3H), 2.77 (dd, J=4.6, 17.6Hz, 1H), 3.42 (dd, J=2.5, 17.6 Hz, 1H), 4.1-4.2 (m, 1H), 4.95-5.1 (m,2H), 7.62 (br d, J=9.2 Hz, 1H), 7.93 (br d, J=9.0 Hz, 2H), 8.18 (d,J=9.0 Hz, 2H), 9.28 (br s, 1H); MS (LSIMS) m/e 425 (46, M⁺ +1), 369(100); [α]_(D) ²⁰ -11.8° (c=1.0, DMF); Analysis calculated for C₁₈ H₂₄N₄ O₈ : C, 50.94; H,5.70; N13.20; found: C, 50.86; H, 5.68; N, 12.85.

D. t-BOCValAlaAsp-p-nitroanilide

t-BOCAlaAsp-p-nitroanilide (424 mg, 1.00 mmole) was dissolved in neatTFA (10 mL) and stirred at room temperature for 1 hour. The solvent wasthen evaporated in vacuo and the residue was then dissolved in 10 mL ofCH₂ Cl₂ which was then evaporated in vacuo. This dissolution with CH₂Cl₂ and subsequent evaporation was repeated two more times giving alight yellow foam. This foam was suspended in CH₂ Cl₂ (10 mL) andtreated with DIEA (129 mg, 1.00 mmole). To this suspension was addedt-BOCVal N-hydroxysuccinimide ester (314 mg, 1.00 mmole), DMF (10 mL)and sufficient additional DIEA to make the mixture neutral. After themixture was stirred at room temperature for 24 hours, the solvents wereremoved in vacuo (high vacuum) and the residue triturated with 1N HCl.The resulting light yellow solid was collected, washed with water anddried. Chromatography (5:35:60--acetic acid:ethyl acetate:hexane to5:55:40--acetic acid:ethyl acetate:hexane) gave 348 mg (66%) of a lightyellow glass that was recrystallized from ethyl acetate to give 128 mgof an amorphous solid: mp 200°-202° C.; ¹ H NMR (DMSO-d₆) δ 0.78 (d,J=6.7 Hz, 3H), 0.81 (d, J=6.8 Hz, 3H), 1.20 (d, J=8.1 Hz, 3H), 1.37 (s,9H), 1.85-1.95 (m, 1H), 2.60 (dd, J=7.5, 16.6 Hz, 1H), 2.76 (dd, J=6.3,16.6 Hz, 1H), 3.75-3.85 (m, 1H), 4.2-4.3 (m, 1H), 4.63 (m, 1H), 6.77 (d,J=8.6 Hz, 1H), 7.88 (d, J=9.2 Hz, 2H), 8.02 (d, J=7.7 Hz, 1H), 8.21 (d,J=9.2 Hz, 2H), 8.41 (d, J=7.0 Hz, 1H), 10.53 (brs 1H); MS (LSIMS) m/e546 (21, M⁺ +Na), 524 (22, M⁺ +1), 507 (12), 468 (45), 424 (57), 330(35), 286 (34), 215 (100); [α]_(D) ²⁰ -27.7° (c=1.0, DMF); Analysiscalculated for C₂₃ H₃₃ N₅ O₉ : C, 52.76; H, 6.35; N, 13.38; found: C,52.45; H, 6.57; N, 12.76.

EXAMPLE 5 AcValAlaAsp-p-nitroanilide

t-BOCValAlaAsp-p-nitroanilide (174 mg, 0.33 mmole) was dissolved in neatTFA (5 mL) and stirred at room temperature for 1 hour. The TFA wasremoved in vacuo and the residue was then dissolved in 5 mL of CH₂ Cl₂which was then evaporated in vacuo. This dissolution with CH₂ Cl₂ andsubsequent evaporation repeated two more times. The residue wasdissolved in dioxane/water (5 mL, 4:1) and treated with acetic acidN-hydroxysuccinimide ester (63 mg, 0.40 mmole) and NaHCO₃ (139 mg, 1.65mmole). After 18 hours, the mixture was diluted with 1N HCl (25 mL) andextracted three times with ethyl acetate. The combined extracts weredried with MgSO₄, filtered and concentrated to a yellow solid which wasrecrystallized from ethyl acetate/ethanol to give 33 mg (21%) of lightyellow powder: mp 196°-200° C. (dec.); ¹ H NMR (DMSO-d₆) δ 0.80 (d,J=6.6 Hz, 3H), 0.82 (d, J=5.1 Hz, 3H), 1.20 (d, J=7.2 Hz, 3H), 1.85 (s,3H), 1.85-2.0 (m, 1H), 2.60 (dd, J=7.5, 16.6 Hz, 1H), 2.75 (dd, J=6.2,16.6 Hz, 1H), 4.1-4.2 (m, 1H), 4.2-4.3 (m, 1H), 4.6-4.7 (m, 1H), 7.8-7.9(m, 3H), 8.11 (d, J=6.8 Hz, 1H), 8.21 (d, J=9.3 Hz, 2H), 8.27 (d, J=7.2Hz, 1H), 10.51 (s, 1H), 12.45 (br s, 1H); MS (LSIMS) m/e 488 (6, M⁺+Na), 466 (23, M⁺ +1), 449 (7) 328 (15), 213 (44), 142 (100); [α]_(D) ²⁰-24.9° (c=1.0, DMF); Analysis calculated for C₂₀ H₂₇ N₅ O₈ : C, 51.60;H, 5.85; N, 15.05; found: C, 50.16; H, 5.79; N, 14.06.

EXAMPLE 6 CBZValAlaAsp-p-nitroanilide

A. CBZAsp(β-t-butyl)-p-(t-BOC amino)anilide

CBZAsp(β-t-butyl)OH dicyclohexylamine salt (2.52 g, 5.00 mmole),4-(t-BOC amino)aniline (1.04 g, 5.00 mmole), DEC.HCl (1.44 g, 7.5mmole), N-hydroxybenzotriazole hydrate (675 mg, 5.0 mmole) and DIEA (323mg, 2.5 mmole) were combined in dry DMF (50 mL) and stirred at roomtemperature for 24 hours. The mixture was diluted with ether (150 mL)and washed twice with 1N HCl, twice with saturated NaHCO₃, and once with1N HCl. After drying over MgSO, filtration and concentration in vacuo,an off-white solid was obtained which was recrystallized fromcyclohexane/ethyl acetate to give 2.17 g (84%) of tan solid. Ananalytical sample was prepared by recrystallization fromhexane/ethylacetate: mp 131°-133° C. (softens 120° C.); ¹ H NMR (CDCl₃)δ 1.43 (s, 9H), 1.51 (s, 9H), 2.67 (dd, J=7.1, 17.2 Hz, 1H), 2.97 (dd,J=4.1, 17.2 Hz, 1H), 4.6-4.7 (m, 1H), 5.16 (s, 2H), 6.10 (br d, 1H),6.45 (br s, 1H), 7.30 (d, J=9.1 Hz, 2H), 7.35-7.45 (m, 7H), 8.41 (b s,1H); MS (LSIMS) m/e 513 (46, M⁺), 457 (40), 402 (38), 358 (40), 243(32), 178 (31), 152 (100); [α]_(D) ²⁰ -19.2° (c=1.0, methanol); HPLCretention time: 4.32 minutes (30%), 17.17 minutes (40%); Analysiscalculated for C₂₇ H₃₅ N₃ O₇ : C, 63.14; H, 6.87; N, 8.18; found: C,63.24; H, 6.94; N, 8.05.

B. CBZAlaAsp(β-t-butyl)-p-(t-BOC amino)anilide

CBZAsp(β-t-butyl)-p-(t-BOC amino)anilide (1.17 g, 2.28 mmole) washydrogenated over 10% Pd-C (120 mg) at 3 atm. in CH₃ OH (20 mL) at roomtemperature for 1 hour. The reaction mixture was filtered through anylon filter and the filtrate concentrated to an oil. This was dissolvedin CH₂ Cl₂ (23 mL) and CBZAla N-hydroxysuccinimide ester (803 mg, 2.51mmole) was added. After being stirred at room temperature for 24 hours,the reaction mixture was washed twice with 1N HCl and twice withsaturated NaHCO₃ and then dried over MgSO₄. Filtration and concentrationin vacuo gave a white solid which was recrystallized fromcyclohexane/ethyl acetate to give 750 mg (56%) of white powder: mp183°-185° C. (with gas evolution); ¹ H NMR (DMSO-d₆) δ 1.19 (d, J=7.2Hz, 3H), 1.35 (s, 9H), 1.45 (s, 9H), 2.54 (dd, J=7.7, 15.6 Hz, 1H), 2.72(dd, J=6.4, 15.6 Hz, 1H), 4.0-4.1 (m, 1H), 4.6-4.7 (m, 1H), 4.99 (d,J=12.5 Hz, 1H), 5.03 (d, J=12.5 Hz, 1H), 7.25-7.4 (m, 7H), 7.48 (d,J=8.9 Hz, 2H), 7.58 (br d, J=6.5 Hz, 1H), 8.23 (br d, J=8.1 Hz, 1H),9.26 (br s, 1H), 9.68 (br s, 1H); MS (LSIMS) m/e 585 (34, M⁺ +1), 584(44, M⁺), 529 (41), 528 (29), 473 (50), 321 (94) 243 (100); [α]_(D) ²⁰-28.9° (c=1.0, methanol); Analysis calculated for C₃₀ H₄₀ N₄ O₈ : C,61.63; H, 6.90; N, 9.58; found: C, 61.66; H, 7.15; N, 9.52.

C. CBZValAlaAsp(β-t-butyl)-p-(t-BOC amino)anilide

CBZAlaAsp(β-t-butyl)-p-(t-BOC amino)anilide (1.97 g, 3.37 mmole) washydrogenated over 10% Pd on carbon (200 mg) at 3 atm. in CH₃ OH (50 mL)at room temperature for 1 hour. The reaction mixture was filteredthrough a nylon filter and the filtrate concentrated to a white gummysolid. This solid was suspended in DMF (12 mL) and CBZValN-hydroxysuccinimide ester (1.29 g, 3.71 mmole) was added. After beingstirred at room temperature for 24 hours, the reaction mixture wasdiluted with a saturated solution of NaHCO₃ and stirred for 15 minutes.The precipitated product was collected, washed with water and driedunder high vacuum to give 2.16 g (94%) of a fine white powder: mp232°-233° C. (with gas evolution); ¹ NMR (DMSO-d₆) δ 0.81 (d, J=8.7 Hz,3H), 0.84 (d, J=6.9 Hz, 3H), 1.19 (d, J=7.0 Hz, 3H), 1.33 (s, 9H),1.85-2.0 (m, 1H), 2.52 (dd, J=7.6, 15.8 Hz, 1H), 2.69 (dd, J=6.4, 15.8Hz, 1H), 3.8-3.9 (m, 1H), 4.2-4.3 (m, 1H), 4.6-4.7 (m, 1H), 5.01 (d,J=12.8 Hz, 1H), 5.03 (d, J=12.8 Hz, 1H), 7.25-7.4 (m, 8H), 7.47 (d,J=9.0 Hz, 2H), 8.08 (br d, J=6.9 Hz, 1H,) 8.21 (br d, J=7.9 Hz, 1H),9.25 (br s, 1H), 9.79 (br s, 1H); MS (LSIMS) m/e 684 (51, M⁺), 420 (40),119 (100); [α]_(D) ²⁰ -16.5° (c=1.0, DMF); Analysis calculated for C₃₅H₄₉ N₅ O₉ : C, 61.47; H, 7.22; N, 10.24; found: C, 61.29; H, 6.93; N,10.20.

D. CBZValAlaAsp-p-nitroanilide

CBZValAlaAsp(β-t-butyl)-p-(t-BOC amino)anilide (900 mg, 1.32 mmole) wasdissolved in cold TFA (13 mL) and stirred at 0° C. for 4 hours. The TFAwas removed in vacuo and acetic acid (26 mL) added to the residue. Tothe resulting suspension was added NaBO₃ (2.03 g, 13.2 mmole) and themixture stirred for 18 hours at room temperature. The reddish-orangereaction mixture was concentrated. Water and ethyl acetate (200 mL) wereadded to the residue and a small amount of 1N HCl added to bring the pHto about 1. The separated ethyl acetate layer was twice washed with 1NHCL and was then dried with MgSO₄, filtered and concentrated to a lightbrown solid. This was absorbed onto silica gel and charged onto acolumn. Elution (2.5:2.5:95--acetic acid:methanol:CH₂ Cl₂) gave 474 mg(64%) of product. Further purification was achieved by preparative thinlayer chromatography (5:5:95--acetic acid:methanol:CH₂ Cl₂): mp204°-206° C. (dec., softens 188° C.); ¹ H NMR (DMSO-d₆) δ 0.80 (d, J=8.1Hz, 3H), 0.83 (d, J=7.0 Hz, 3H), 1.21 (d, J=7.1 Hz, 3H), 1.85-2.0 (m,1H), 2.61 (dd, J=7.7, 16.6 Hz, 1H), 2.77 (dd, J=6.0, 16.6 Hz, 1H),3.8-3.9 (m, 1H), 4.2-4.3 (m, 1H), 4.6-4.7 (m, 1H), 5.02 (s, 2H),7.25-7.4 (m, 6H), 7.88 (d, J=9.3 Hz, 2H), 8.12 (d, J=6.6 Hz, 1H), 8.21(d, J=9.3 Hz, 2H), 8.38 (d, J=7.3 Hz, 1H), 10.48 (s, 1H), 12.40 (br s,1H); MS (LSIMS) m/e 580 (M⁺ +Na, 17) 558 (M⁺ +1, 71), 541 (19), 527(32),420 (51), 305 (100); [α]_(D) ²⁰ -23.9° (c=1.0, DMF); Analysis calculatedfor C₂₆ H₃₁ N₅ O₉.0.25H₂ O:C, 55.56; H, 5.65 N, 12.46; found: C, 55.33;H, 5.56; N, 12.09.

EXAMPLE 7 Assay

The use of the compounds of this invention in identifying inhibitors ofinterleukin 1β converting enzyme (ICE) and, consequently, demonstratingthe latter compounds' effectiveness for treating inflammatory diseasesis disclosed by the following in vitro assay. Other procedures forpurification and assaying ICE are disclosed in Black et al., FEBSLetters, 247, 386-390 (1989), and Thornberry et al., Nature, 356,768-774 (1992).

Cell Culture and Lysates

Human monocyte cell line, THP-1 (ATCC-TIB 202) was grown in RPMI media1640 (Gibco BRL Gaithersburg, Md. 20877) with 10% fetal bovine serum,harvested by centrifugation, washed twice in Dulbecco's PBSdithiothreitol without Ca⁺⁺, and resuspended in 10 mM Tris-HCl pH 8buffer containing 5 mM DTT (dithiothreitol), 1 mM EDTA (ethylene diaminetetraacetic acid), 1 mM PMSF (phenylmethyl sulfonylfluoride), 1 μg/mlpepstatin, and 1 μg/ml leupeptin at 1-3×10⁸ cells per ml. Cells werefrozen at -70° C. until use and then lysed by thawing. Lysates werecleared by centrifugation at 20,000×g for 1 hour followed by 120,000×gfor 1 hour.

Partial Purification of ICE Activity by Ion-Exchange Chromatography

ICE activity was purified from THP-1 cell lysates by threechromatographic steps: (A) Thp-1 cell lysate (1.5 L) was desalted by G25column chromatography (Pharmacia LKB Biotechnology) (B) The proteinfraction was then subjected to ion-exchange chromatography onQ-Sepharose Fast Flow (Pharmacia LKB Biotechnology) in buffer A (20 mMTris pH 7.8 containing 5 mM EDTA, 1 mM PMSF, 1 μg/ml pepstatin, and 1μg/ml leupeptin). ICE activity was eluted with a gradient of NaCl inbuffer A. (C) The active fractions from B were desalted, concentratedand subjected to MonoQ (trademark) (Pharmacia LKB Biotechnology) columnchromatography. ICE activity was then eluted in a NaCl gradient. ActiveICE fractions from C were pooled and used to bind immunoaffinity beadscontaining covalently linked antibodies raised against the first 11N-terminal residues of ICE (NH₂-Asp-Pro-Ala-Met-Pro-Thr-Ser-Ser-Val-Lys-Leu-Cys-CONH₂).

Immobilization of ICE

Immobilization of ICE to immunoaffinity beads was done followingstandard protocols. Briefly, IgG fractions were covalently linked toprotein A beads as described by the manufacturers (Antibody OrientationKit Protein A Agarose supplied by Affinica (trademark) (Productmanufactured by Schleicher and Schuell)). Protein A beads were pelletedby centrifugation and washed with 5 times their volume with Affinica(trademark) supplied "binding buffer". IgG, appropriately diluted in"binding buffer," was then bound to the beads. The beads were thenwashed and the bound IgG was covalently linked with dimethylsuberimidate. After the reaction was stopped with the "quenching buffer"supplied by Affinica (trademark), the immunoaffinity beads were washedand stored in PBS buffer containing 0.02% NaN₃. To bind theimmunoaffinity beads with MonoQ purified ICE preparations, the beadswere washed in 10 mM Tris HCL buffer pH 7.8 containing 5 mM DDT, 1 mMEDTA-NA, 1 ug/ml peptstatin, 1 ug/ml leupeptin and 10% glycerol("washing buffer"). After the wash, the beads were mixed with ICE in thepresence of 10% DMSO final concentration. The suspension was rotatedslowly for 1 hour at room temperature. Subsequently, the beads werethoroughly washed with the "washing buffer" before they were used andresuspended in an equal volume of this buffer in the enzymatic assay.

Assay Procedure

The enzymatic reaction was carded out at 25° C. in 96 microtiter wells(100 μl final volume) with active enzyme immobilized to anti-ICEantibodies covalently linked to protein A beads as described above. Theincubation mixtures for the enzymatic assays contained 0.01 to 1.5 mM ofthe pNA substrates and was made up in 8.35 mM MES, 4.17 mM Tris, 4.17 mMacetic acid, 4.6M DMSO, 0.8 mM EDTA and 4.17 mM DTT (finalconcentrations) adjusted to a final pH of 7.

The enzymatic reaction was monitored spectrophotometrically at 405 nM.The increase in absorbance at this wave length resulted from the releaseof pNA chromophore after hydrolysis by ICE of the peptidic-pNAsubstrate. The release of the chormophore was linear with reaction timeand the rates observed were proportional to ICE and substrateconcentration. The spectrophotometric assay greatly facilitated thequantitative determination of kinetic constants and assessment of theenzyme specificity (Vmax/Km). In addition, comparison of inhibitorycompounds was possible. These compounds could easily be introduced inthe assay and compared as to the type (competitive, uncompetitive,mixed) and degree (Ki) of inhibition they can effect on catalysis (SeeA. Cornish-Bowden, Fundamentals of Enzyme Kinetics, Butterworth and Co.,Ltd., London (1979).

The rates, kinetic constants and relative specificity for the pNAsubstrates for ICE are presented in Table 1. The data are consistentwith the interpretation that tripeptides and tetrapeptides aresubstrates for this enzyme, compounds smaller than tripeptides are not.In addition two known peptidic compounds (prepared by a solid phasepeptide synthesizer) known to be hydrolyzed by ICE, p70(H-Asn-Glu-Ala-Tyr-Val-His-Asp-Ala-Pro-Val-Arg-Ser-Leu-Asn and p48(Ac-Tyr-Val-His-Asp-Ala-NH₂), were also found to behave as competitiveinhibitors of the hydrolysis of CBZ-Val-Ala-Asp-pNA by affecting Km butnot Vmax (Ki(mM) was found to be 0.46 mM and 1.6 mM respectively). Thisdemonstrated the ability of the assay to quantitatively evaluatepotential inhibitors of ICE.

                  TABLE 1                                                         ______________________________________                                        Evaluation of pNA Substrates                                                  Com-   Increase In         mOD/                                               pound  Absorbance          min         Relative                               of     Rates       Km      Vmax  Vmax/ Vmax/                                  Example                                                                              (mOD/min)   (mM)    (Rates)                                                                             Km    Km                                     ______________________________________                                        4      0.17    0.10    0.443 0.24  0.54  1.00                                 5      0.15    0.08    0.447 0.25  0.56  1.04                                 6      0.18    0.15    0.0979                                                                              0.21  2.15  3.98                                 1      0.21    0.17    0.0438                                                                              0.27  6.16  11.41                                ______________________________________                                    

We claim:
 1. A method of detecting inhibitors of interleukin 1βconverting enzyme (ICE) comprising evaluating a compound's capacity toinhibit the ICE-induced hydrolysis of a para-nitroanilide peptideselected from the group consisting of PhCH₂ CH₂COValAlaAsp-p-nitroanilide, PhCH₂ NHCoValAlaAsp-p-nitroanilide,t-BOCValAlaAsp-p-nitroanilide, AcValAlaAsp-p-nitroanilide andCBZValAlaAsp-p-nitroanilide by:a) mixing active ICE immobilized toanti-ICE antibodies covalently linked to protein A beads with saidpara-nitroanilide peptide in the presence or absence of said compound ina reaction vessel, and b) monitoring enzymatic activityspectrophotometrically at 405 nm.